Biophysical analysis of mutant proteins. (a) Representative chromatograms from analytical size-exclusion columns of VP35 IID mutants R312A (blue), K319A/R322A (red) and K339A (green). The inset shows a Coomassie-stained SDS–PAGE gel of the highly purified final protein samples used in the crystallization trials. Sample order (from left to right) is R312A, K319A/R322A and K339A. (b) Normalized fluorescence emission data for ThermoFluor assays of VP35 IID mutants. Curves are colored as in (a). (c) The derivatives of the data in (b) show differences in Tm values for mutant proteins. The Tm values for R312A, K319A/R322A and K339A are 330 ± 0.2, 325.3 ± 0.2 and 323.0 ± 0.7 K, respectively. Curves are colored as in (a).